@article{mbs:/content/journal/micro/10.1099/00221287-147-8-2077, author = "Matsuoka, Masayoshi and Takahama, Kazutaka and Ogawa, Takahira", title = "Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers", journal= "Microbiology", year = "2001", volume = "147", number = "8", pages = "2077-2087", doi = "https://doi.org/10.1099/00221287-147-8-2077", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-147-8-2077", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", keywords = "merodiploid", keywords = "Kmr, kanamycin-resistant", keywords = "Strr, streptomycin-resistant (chromosomal)", keywords = "Apr, ampicillin-resistant", keywords = "Cms, chloramphenicol-sensitive", keywords = "Synechococcus", keywords = "gene conversion", keywords = "psbAI", keywords = "Kms, kanamycin-sensitive", keywords = "Cmr, chloramphenicol-resistant", keywords = "homologous recombination", keywords = "Smr, streptomycin-resistant", keywords = "Strs, streptomycin-sensitive (chromosomal)", abstract = "Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile gene replacement system without this restriction is reported in a unicellular cyanobacterium, Synechococcus sp. PCC 7942. The system is based upon the dominance of a streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with a Lys-43→→→Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps12 gene and a kan gene conferring kanamycin resistance was integrated into the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite spontaneous gene conversion in these merodiploids to produce streptomycin-resistant progeny at frequencies ranging from 1×10−5 to 5×10−5, homologous recombination could be induced by transforming the merodiploids with template plasmids carrying psbAI 5′ and 3′ non-coding sequences flanking the D1 coding sequence, which was then replaced by either the gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3–16% of streptomycin-resistant progeny of the merodiploids after transformation were homogenote recombinants with concomitant loss of the kan gene, even in these polyploid cyanobacteria. The rps12-mediated gene replacement thus makes it possible to construct mutants free from drug resistance markers and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.", }